1. Field of the Invention
In general, the present invention relates to assay methods and devices for the determination of the presence or amount of an analyte in a test sample. In particular, the invention relates to novel binding assay devices which include a means to confirm the assay result.
2. Description of Related Art
In the development of the medical diagnostics field, there has been explosive growth in the number of substances to be detected in physiological test samples. Various analytical procedures are commonly used in diagnostic assays to determine the presence and/or amount of these substances of interest or clinical significance. These clinically significant or interesting substances are referred to as analytes. Diagnostic assays have become an indispensable means for detecting analytes in test samples, and for the most part the medical profession has used highly automated clinical laboratories and sophisticated equipment for these determinations.
The ability to use materials which specifically bind to an analyte of interest has created a need for diagnostic devices based on the use of binding assays. Binding assays incorporate specific binding members, typified by antibody and antigen immunoreactants, wherein one member of the specific binding pair is labeled with a signal-producing compound (e.g., an antibody labeled with an enzyme, a fluorescent compound, a chemiluminescent compound, a radioactive isotope, a direct visual label, etc.). For example, in a binding assay the test sample suspected of containing analyte can be mixed with a labeled reagent, e.g., labeled anti-analyte antibody, and incubated for a period of time sufficient for the immunoreaction to occur. The reaction mixture is subsequently analyzed to detect either that label which is associated with an analyte/labeled reagent complex (bound labeled reagent) or that label which is not complexed with analyte (free labeled reagent). As a result, the amount of free or bound label can be correlated to the amount of analyte in the test sample.
The solid phase assay format is a commonly used binding assay technique. There are a number of assay devices and procedures wherein the presence of an analyte is indicated by the analyte's binding to a labeled reagent and an immobilized or insoluble complementary binding member. The immobilized binding member is bound, or becomes bound during the assay, to a solid phase such as a dipstick, teststrip, flow-through pad, paper, fiber matrix or other suitable solid phase material. The binding reaction between the analyte and the assay reagents results in a distribution of the labeled reagent between that which is immobilized upon the solid phase and that which remains free. The presence or amount of analyte in a test sample is typically indicated by the extent to which the labeled reagent becomes immobilized upon the solid phase material.
The use of reagent-impregnated teststrips in specific binding assays is well-known. In such procedures, a test sample is applied to one portion of the teststrip and is allowed to migrate or wick through the strip material. Thus, the analyte to be detected or measured passes through or along the material, possibly with the aid of an eluting solvent which can be the test sample itself or a separately added solution. The analyte migrates into a capture or detection zone on the teststrip, wherein a complementary binding member to the analyte is immobilized. The extent to which the analyte becomes bound in the detection zone can be determined with the aid of the labeled reagent which can also be incorporated in the teststrip or which can be applied separately.
An early teststrip device is described by Deutsch, et al. in U.S. Pat. No. 4,361,537. In general, the device comprises a material capable of transporting a solution by capillary action, i.e., a wicking or chromatographic action. Different areas or zones in the teststrip contain the assay reagents needed to produce a detectable signal as the analyte is transported to or through such zones. The device is suited for both chemical assays and binding assays and uses a developer solution to transport analyte along the strip.
Many alternatives to or variations on the Deutsch, et al. device have been disclosed. For example, Grubb, et al. (U.S. Pat. No. 4,168,146) describe the use of a porous teststrip material to which is covalently bound an antigen-specific antibody. In performing the assay, the teststrip is immersed in a solution suspected of containing an antigen, and capillary migration of the solution through the teststrip is allowed to occur. As the antigen moves through the teststrip it binds to the immobilized antigen-specific antibody. The presence of antigen is then determined by wetting the teststrip with a second antigen-specific antibody to which a fluorescent or enzyme label is covalently bound. Quantitative testing can be achieved by measuring the length of the strip that contains bound and labeled antigen.
Weng, et al. (U.S. Pat. No. 4,740,468) describe another device and method for performing a specific binding assay. The assay involves both an immobile second binding member which binds to a mobile first binding member and an immobilized analog of the analyte which removes unbound first binding member from the assay system prior to its contacting the detection site. Greenquist, et al. (U.S. Pat. No. 4,806,311) describe a similar device wherein a first immobilized reagent, such as an analyte-analog, is present in a reagent zone to remove free monovalent labeled-binding members from the assay system prior to the test samples contact with the detection layer reagents.
Analyte detection in a specific binding assay can be achieved using either a sandwich assay or competitive assay format. The confirmation of the assay result is typically accomplished by treating the test sample to neutralize the analyte and then repeating the assay. This process involves several additional procedural steps and reagent additions by the user and subjects the assays to an increased risk of error. If the initial assay result is not confirmed, the initial positive result may be falsely positive, thereby leading to errors in diagnosis and treatment. Thus, there is a need for a specific binding assay format and device which permit the simultaneous confirmation of the assay result without the need for additional operations.